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1979

CELL PROLIFERATION AND COLLAGEN SYNTHESIS IN EXPLANT CELL PROLIFERATION AND COLLAGEN SYNTHESIS IN EXPLANT

CULTURES OF ARTERIOSCLEROTIC RABBIT AORTA CULTURES OF ARTERIOSCLEROTIC RABBIT AORTA

Douglas Otto Fisher University of Rhode Island

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CELL PR0LIFERATION AND COLi.J:.GEN SYNTHES IS IN:

EXPLANT CULTURES OF ARTERIOSCLEROTIC RABBIT AORTi\

BY

DOUGLAS OTTO FISHER

A THESIS SUBMITTED I N PARTIAI. FULFILL~1ENT OF T3£

REQUIREMENTS FOR THE DEG!IBE OF

DOCTOR OF PHILOSOPHY

IN

PHARM_~CEUTICAL SCIENCES

(PHARMACOLOGY AND TOXICOLOGY)

UN IVERSITY Of' RHODE I SLA_m

19 79

DOCTOR OF PHILOSOPHY DISSERTATION

OF

DOUGLAS OTTO FISHER

Approved:

Dissertation Committee

Dean of the Graduate School

UNIVERSITY OF RHODE ISLAND

197 9

ABSTRACT

Fisher, Douglas Otto. Ph.D., University of Rhode Island. 1979. Cell Proliferation and Collagen Synthesis in Explant Cultures of Arteriosclerotic Rabbit Aorta. Major Professor: Dr. George c. Fuller.

The rate of cell proliferation and the ratio of type I/

III collagen s ynthesized by aorta organ culture and cultured

smooth muscle cel l s (SMC) was determined in cells derived

from rabbits with injury induced (daily epinephrine and thy-

roxine administration) arteriosclerosis and increased va.scu-

lar collagen s ynthesis. Tissue was taken from medial smooth

muscle of normal and diseased rabbits and incubated in organ

or cell culture systems with 2,3- 3H-proline, ascorbate and·

beta-aminopropionitrile. Collagen types were separated by

SDS-polyacrylamide electrophoresis and CMC-chromatography

and quantitated radiometrically. 1icroscopic observation

at 12 days in culture and 3H-thyrnidine in~orporation at 18

days in culture indicated a greater rate of proliferation

of SMC from explants of arterioschlerotic tissue compared

to control. In organ culture the ratio of collagen type

III : type I was 1 : 1 in the control group and 1 : 1.7 for

the arteriosclero.tic group . Collagen type III : type I for

daughter cells at the end of the 4th passage was 1 : 1.4

and 1 : 2.0 for control and ar~eriosclerotic, respectively .

By the 10th passage in culture SMC in both groups were syn-

thesizing almost exclusively type I collagen. This study

indicates that cells from arteriosclerotic smooth muscle ere

fundamentally unique since the ratio of collagen t ype is con­

trolled by gene expression. This property affecting collagen

deposition may play a role in t he pathogenesis of vascular

disease.

ACKNOWLEDGEMENTS

The author wishes to express his appreciation to

Dr. George c. Fuller for guidance and encouragement through-

out this investigation. Thanks are also conveyed to Dr.

David DeFanti for his contributions in support of my gradu­

ate training.

Gratitude is extended to Mrs. Lynne Matoney and Mrs.

Nadylis Wood for their generous technical assistance at

various stages of this investigation. Also acknowledged

is Mr. Ronald Goldberg for his expertise in gel electrophor­

esis.

The author extends sincere appreciation to his wife,

Kathy, for her continued encouragement and understanding

during his tenure in graduate school.

Financial support for this inve3tigation was provided

by AHA #75-670 and NIH #HL16904 grants which were awarded

to George C. Fuller.

iv

ABSTRACT. . . ACKNOWLEDGEMENTS. TABLE OF CONTENTS

LIST OF TABLES. LIST OF FIGURES

INTRODUCTION.

LITERATURE SURVEY

TABLE OF CONTENTS

The Vascular Connective Tissue Matrix

Response of the Vasculature to Injury

The Role of the Smooth Muscle Cell in Arterio-sclerosis .

ii iv

v

vii ·viii

1

4

4

12

13

The Smooth Muscle Cell in Culture 15

Cell Transformation and the Collagen Phenotype. 17

Collagen Heterogeneity: Pathological Implications .

EXPERIMENTAL .

Animals .

Materials

Epinephrine-Thyroxine Induced Arteriosclerosis.

Organ Culture Sy stem for Medial Tissue Explants

Culture of Vascular Smooth Muscle Cells ..

3H-thymidine Incorporation by Explant Cultures.

Electrophoretic Separation of Collagen Types .

CMC-Chromatoqraphy of Collagen Types.

Quantitation of Collagen Types

Statistical _1ethods . .

v

20

23

23

23

24

25

26

27

28

29

29

31

RESULTS. .

Vascular Smooth Muscle Cell Proliferation.

Collagen Synthesis inPrimary Organ Culture

Collagen Synthesis by Vascular SMooth Muscle Cells in Culture . . . . . ..... .

Collagen Heterogeneity by CMC-Chromatography

DISCUSSION

CONCLUSIONS.

P.EFERENCES

VITA

vi

PAGE

33

34

• 36

43

48

54

64

• 6 6

75

Table

1.

2.

3.

4.

5 .

LIST OF TABLES

Estimation of New Cell Growth from M8dial Explants.

3H-thymidine Incorporation by Primary Cultures of Medial Smooth Muscle Cells from Control and Arteriosclerotic Rabbits.

Rate of Synthesis in Organ Culture of 3H­proline Labelled Collagen by Aorta Medial Smooth Muscle from Control and ARteriosclero­tic Rabbits .

3 Rate of Synthesis of H-proline Labelled Collagen by Cultured Smooth Muscle Cells De­rived from Aorta Media of Control and Arteriosclerotic ~abbits.

Comparison of Electrophoretic and Chromato­graphic Results for the Synthesis of Type I and Type III Collagens.

vii

Page

37

42

44

46

52

LIST OF FIGURES

Figure Page

1. Peripheral Outgrowth of Smooth Muscle Cells from Rabbit Aorta ~ledial Explants in Culture. 35

2. Migration of Type I and Type III Collagen Stand­ards on Polyacrylarnide l,els Before and After Reduction with 2-Mercaptoethanol. 39

3. Effect of 2-Mercaptoethanol and Bacterial Collag­enase Treatment on the Electrophoretic Migration of 3H-proline Labelled Collagen Synthesized in Culture by Rabbit Vascular Smooth Muscle Cells .. 40

4. Effect of Age in Culture on the Sy nthesis of Type I and Type III Collagens by Aorta Medial Smooth Muscle Cells from Control and Arterio -sclerotic Rabbits . 47

5. CMC - Chromatography of Labelled Collagen Synthe­sized in Organ Culture by Aorta Medial Smooth Muscle from Control and Arteriosclerotic Rabbits . 50

6. CMC-Chromatography of Labelled Collagen Synthe­sized by Cultured Smooth Muscle Cells Derived from Aorta Media of Control and Arteriosclerotic Rabbits . 51

v iii

INTRODUCTION

Arteriosclerosis is a complex disease involving many in­

trinsic and extrinsic factors which influence the suscepti­

bility of the vasculature to insult and the subsequent clini-

cal sequelae. In arteriosclerosis a variable combination of

metabolic changes seen in the intima and media of arteries

is characterized by" .... the focal accumulation of modified

smooth muscle cells, lipids, complex carbohydrates, blood and

blood products, fibrous tissue and calcium deposits" (World

Health Organization, 1958). These changes predominate in the

major arteries and resistance vessels and by compromising

blood flow, account for the clinical appearance of heart at­

tacks, strokes, senility and memory loss, angina and im­

paired peripheral circulation.

The major current theories on the genesis of arterio­

scl~rosis share the belief that the lesions begin as local­

ized foci of hyperactive smooth muscle cells in the intima

and medial regions of the blood vessels. Authors from this

laboratory have reported evidence, using various techniques,

which establish the existence of increased collagen synthe­

sis in experimentally induced plaques in animals. The

deposition of collagen in the arterial intima is largely re­

sponsible for the occlusive and irreversible nature of

arteriosclerosis (Benditt, 1977). It is believed that the

2

defect in collagen metabolism which precedes the formation of

fibrous vascular plaques is due to a change in the growth

characteristic and function of smooth muscle cells, the pre­

dominant cell type in the arteriosclerotic intima.

Four genetically distinct types of collagen are current­

ly recognized and, although their distribution is tissue

specific, various disease processes and aging can be corre­

lated to altered distribution of collagen type. For example,

type I collagen is the predominant extractable collagen spe­

cies in human fibrous atheromatous plaques whereas type III

appears to be the major aortic medial collagen (Mccullagh

and Balian, 1975). Little is known about the control of

collagen heterogeneity but it could be proposed that a change

in collagen gene expression, as a result of smooth muscle

cell transformation, may be associated with the development

of arteriosclerosis.

This investigation was conducted to examine the hypo­

thesis that the arteriosclerotic lesion in rabbits is asso­

ciated with a distinct population of smooth muscle cells

with altered growth characteristics and function, suggestive

of cellular transformation. To examine this hypothesis the

growth characteristic of cells from primary cultures of con­

trol and arteriosclerotic rabbit aorta were examined. Also,

studies were conducted to determine if daughter cells derived

from medial tissue explants of arteriosclerotic plaques

continue to synthesize a similarly abnormal ratio of type I

3

to type III collagen compared to smooth muscle cells derived

from explants of normal aorta. It is believed that these

studies are valuable to gaining an understanding of the nor­

mal regulation of collagen synthesis and perhaps in screening

for useful drugs in the treatment of abnormalities in collagen

metabolism which occur in various disease states.

4

LITERATURE SURVEY

The Vascular Connective Tissue Matrix

The intercellular matrix of the arterial wall contains

four major types of macromolecules (collagen , glycosamino ­

glycans, elastin and glycoproteins) . Most chemical studies

have been carried out using the aortas of different animal

species; only limited information is available on other ar ­

teries or veins. Several reviews discuss the chemical and

biological characteristics of these macromolecules (Balazs,

1970; Robert, 1970; Slavkin, 1972). Much of the early knowl­

edge on the chemistry and biosynthesis of elastin was gained

from experimental work by Partridge (1970, 1972). Several

laboratories have studied the composition and biosynthesis

of glycosaminoglycans and glycoproteins in normal and patho­

logical arterial walls (Engel, 1971; Kumar et al., 1967;

Moczar and Robert, 1970; Srinivasan et al., 1971). Consid­

erable interest in arterial collagen has been generated by

research which demonstrated its dynamic role in experimental

atherosclerosis (Levene, 1962; Smith, 1965; Fuller et al.,

1970, i972, 1973, 1976; Crossley et al., 1972; Ooshima et

al., 1974 ) . As a result various laboratories began i so l at­

ing and characterizing aortic collagen genetic types (Chung

et al., 1974; Epstein et al., 1975; Trelstad, 1974; Mc­

cullagh and Balian, 1975).

Collagen synthesis occurs in a series of sequential

steps consisting of assembly of a praline-rich and lysine­

rich polypeptide precursor of collagen (procollagen alpha

chains} enzymatic hydroxylation of some of the prolyl and

lysyl residues and glycosylation of some of the hydroxy­

lysyl residues. The cellular processing of the procollagen

alpha chains also includes formation of the triple helix

with stability provided by disulfide bonds in the nonheli­

cal regions of the molecule (Bornstein and Ehrlich, 1973).

Following secretion, these nonhelical regions are removed

by the extracellular enzyme procollagen peptidase (Lapiere

et al., 1971; Goldberg , et al., 1975). The crosslinking of

collagen, which imparts stability to helical and .fibrillar

collagen, occurs through further extracellular processing

5

by the enzyme lysyl oxidase which oxidatively deaminates

specific lysine residues leaving aldehyde moieties which form

cross links through condensation reactions (Siegel et al.,

1970) .

Data reported by several laboratories, during recent

years, reflects some inconsistency in expressing collagen

content. Factors which introduce variability include:

species, pathological condition, age, method of isolation

and anatomical region. The experimental studies also indi­

cated that a determination of total content as well as con­

centrations is necessary for a correct evaluation of the

metabolic alterations of the macromolecules in studies on

6

vascular injury (Helin et al., 1971). In a recent study of

canine atherosclerosis the percentage of protein synthesis

represented by collagen rose from a mean of less than 5 % in

normal aortic branch arteries to 14 % in severely atheroscler­

otic branch arteries, showing that the increases in collagen

synthesis were not associated simply with an overall in­

crease in protein synthesis (McCullagh and Ehrhart 1974).

Langner and Fuller (1973), reported increased total collagen

and 0.45M NaCl soluble collagen in thoracic aorta of rabbits

with epinephrine-thyroxine induced arteriosclerosis. The

collagen content of an apparently normal human coronary ar­

tery approximates 25 mg/100 mg of dry, defatted tissue

(Tamai et al., 1978). In arteriosclerosis the wet weight

of a vessel increases and therefore content of vascular com­

ponents can be misleading when expressed on the basis of

organ wet weight (Tammi et al., 1978) . In another study

collagen was reported to comprise 30 % of the dry weight of

human fibrous atherosclerotic plaques (Levene and Poole,

1962).

Aortic prolyl hydroxylase has been used as a marker of

collagen synthesis (Fuller and Langner , 1970; Fuller et al.,

1972) and has been shown to increase up to six-fold in an

experimental model of arteriosclerosis in rabbits and in

aorta of miniature pigs fed a lipid-rich diet. Kinetic

properties of this enzyme and the existence of immunological­

ly cross-reacting forms of the prolyl hydroxylase have been

7

investigated for their potential role in regulating the rate

of collagen synthesis (Fuller et al., 1976; Ooshima et al.,

1974). The Vmax of prolyl hydroxylase in an organ affected

by arteriosclerosis increased four-fold, compared to controls,

while the Km remained unchanged (Fuller et al., 1976). Vari­

ous parameters of collagen synthesis including inununological­

ly related protein are elevated in rats with hypertension

induced by deoxycorticosterone acetate-salt (Ooshima et al .,

1974).

During the mid-seventies advancements in connective

tissue methodology led to the separation and ident ification

of three distinct species of collagen (gene products) from

human aorta (Trelstad , 1974). These included principally

type I and III collagens, along with small amounts of type

IV which is associated with basement membranes. Type I

collagen is composed of two, c:<l chains and one, ~2 chain

and designated by the nomenclature as [~l(I)J 2~2. Type III

collagen i s composed of three identical ~l(III) chains,

designated [~l(III)J 3 and has the distinction of containing

disulfide bonds between the o<-chains. It should also be

mentioned that type II collagen is found in cartilage and

invertebral disc and is represented according to the nomen­

clature as [o<l (II)J 3 . Therefore, a total of four genetically

distinct types of collagen have been isolated and character­

ized by modifications of standard electrophoretic or chroma­

tographic methods (Miller et al., 1 971 ) . There are at least

five structural genes involved in the synthesis of the dif­

ferent types and the transcription of these various genes

seems to be tissue specific.

Mccullagh and Belian (1975 ) have suggested that in ad­

dition to the increased accumulation of collagen reported

8

in arteriosclerosis an alteration in the ratio of collagen

types may have important implications. Their investigations

using pepsin extracted collagen from blood vessels indicated

the composition was approximately 70% type III and 30% type

I. In contrast, collagen extracted from atherosclerotic

blood vessels contained 35% type III and 65% ty~e I.

Another component of the vascular connective tissue

matrix is the glycosaminoglycans, which include hyaluronic

acid, heparin sulfate, chondroitin 4-sulfate, chondroitin

6-sulfate and dernatan sulfate. These macromolecules, which

occur linked to proteins, are composed of repeating units

of substituted hexoses, such as N-ace tyl-D-glucosamine.

Some of them carry the distinction of being sulfated. The

glycosaminoglycans (GAGs) are jellylike, sticky or slippery

substances that provide intercellular lubrication and act

as a flexible cement, New knowledge is available concern­

ing the structure and biosynthesis of these substances in

the aorta of several species. The methods used in current

investigations i nvolve separation and fractionation by

digestion with hyaluronidase and CFC-cellulose micro­

chromatography lThunnel, 1967 ) . The GAG fractions can be

9

quantitated by measuring hexosamine content (Boas, 19 3 3 ) .

Great variations between species have been reported for both

total GAGs per mg. dry aorta or mg . DNA and in the relative

distribution of different GAGs lEngel, 1971 ) , Reports on

the changes of these substances in human arteriosclerosis

do not completely agree (Helin et al., 1970} (Tammi et al.,

1978), However , most agree that chondroitin 4-6 sulfate is

the principle GAG in the large .human arteries .

The interactions of collagen with glycosaminoglycans

has been a subject of investigation for a number of years

(Mathews, 19651. By electron microscopy a regularly distri­

buted parallel or transverse alignment of the. GAG molecule,

along the collagen fibril, has been visualized (Serafini­

Francassini et al,, 19701. Ruthenii.l.ffi red-positive fila­

ments interconnecting collagen fibers have been demonstrated

in rat aorta (Kajikawa , 1970) . Lysine and hydroxylysine

residues as well as the glycosidically bound carbohydrates

in colla~en participate in the interactions between colla­

gen and GAGs. Ionic interactions as well as hydrogen-bond

formation could be significant contributors to the integrity

of connective tissues. It is quite likely that highly

glycosylated collagen, like type II, is most dependent on

such types of interactions, particularly since it is found

in areas rich in glycosaminoglycans l~imni, 1974 1 .

Similar to the glycosaminoglycans which occur linked

to proteins, the glycoproteins are complex pol y saccharides

10

found linked to lipids as well as protein . A large number of

proteins can be classed as glycoproteins because of the pre ­

sence of an oligosaccharide moiety . The term glycoprotein

is frequently restricted to those proteins that contain small

amounts of carbohydrate, less than 4 % hexosamine . Seven

sugars account for almost all the carbohydrate residues in

mammalian glycoproteins. With the exception of L-fucose, the

other suga~s are of the D configuration : N-acetylglucosa­

mine, N-acetylgalacto samine, galactose, mannose, sialic acid

and glucose. The last (glucose) occurs only in proteins of

the collagen type where hydroxylysine is linked to galac­

tose by a glycosidic bond.

Glycoproteins have been extracted by several authors

from aortic tissue and some of the glycoproteins were puri­

fied and characterized (Moczar and Robert, 1970; Moczar

et al., 1970). An elevated level of glycoproteins was re­

ported in arteriosclerotic intima by Hullinger and Manley

(1969) , confirming preceding studies by Nakamura (1968) and

Schoenbeck et al. (1962) . Srinivasan (1971) reported the

presence of tissue specific glycoproteins in several connec­

tive tissues, including aorta. Similar structural glyco­

proteins were isolated from elephant aorta by 1cCullagh

et al. (1973). It was shown that elephant glycoproteins

cross-react with antibody raised against human aorta struc­

tural glycoproteins. It was suggested that there is an

organ-specific immunochemical pattern for these substances.

11

This evidence was consistent with an earlier proposition that

structural glycoproteins could represent the primitive

(phylogenetically) intercellular macromolecular meshwork on­

to which the more elaborate collagen and e lastin fibers are

deposited.

Elastin is the most specific and conspicuous macromo­

lecular component of the intercellular matrix of young large

arteries, such as aorta. The amino acid composition of

elastin, established by Partridge (1 963) strongly suggests

that hydrophobic interactions provide the stabilizing forces

responsible for its tertiary and quarternary structure.

Robert and Poullain (19 64), proposed the hydrophobic hypo­

thesis of elastin elasticity which has been confirmed by

more recent findings (Hoffman, 1971) . It is not within the

scope of this literature survey to thoroughly address the

most current investigations on the physiochemical proper­

ties and biosynthesis of elastin (see Ross and Bornstein,

1969; Abraham et al., 1975). However, it would be appropri­

ate to stress that the fragmentation-degradation of elastic

fibers and laminae is one of the most conspicuous findings

of the atherosclerotic changes in aortas. This is observed

in the human atherosclerotic plaque and it seems to be in­

dependent of the method used to induce experimental arterio­

sclerosis. Neutrophil granulocytes (Janoff , 1970) and blood

platelets were shown to contain proteases with elastolytic

activity (Legrand et al., 1973) and it is quite probable

12

that similar enzymes may be isolated from aorta, where the

degradation of elastin during atherosclerosis has been demon­

strated.

Response of the Vasculature to In~

The repair processes have been proposed to be character­

istic features in various vascular diseases, among these

arteriosclerosis and vasculitis (Helin et al., 1972). _1echan­

ical strain and the subsequent inflammation has been shmm to

be an important initiating factor in the development of

aortic arteriosclerosis in rabbits subjected to systemic hy­

poxia and intravenous injections of catecholamine (Helin and

Lorenzen, 1969; Crossley et al., 1972). The investigations

by these and other authors have established significant re­

lationships between vascular repair following injury and

atherogenesis (Helin et al . , 1971; Hartman, 1977; Kobaysi,

1969). The nature of the repair and the ultimate outcome

of the entire process depends, among other factors, upon

the characteristics of the injurious agent, duration of ex­

posure to it, the type of initial local manifestations and

mural reactions and the status of the host. Owing to the

peculiarities of structure and function, the arterial wall

has limited versatility in defense mechanisms and the de­

fense forces are hampered not only by the lack of mural

capillaries but also by the fact that the artery is never

at rest and thus an important healing-promoting factor is

absent.

13

A number of the established processes in inflammation

and repair are operative following vascular injury and have

been reviewed in detail (Robbins, 1974). Two tissue reac­

tions are characteristically obse~ved in atherosc~erosis and

appear to be unique for this disease process . One is the

regressive change of intimal smooth muscle cells with lipid

accumulation representing fatty metamorphosis. Smooth

muscle cells elsewhere in the body are not known to undergo

fatty metamorphosis. The other unusual feature in athero-

sclerosis is t~e formation of the repair tissue that is

avascular but contains modified smooth muscle cells which

are responsible entirely for the organization and connect­

ive tissue elaboration. These two events give rise to

progressive diffuse intimal thickening and fibrous plaque

formation which are the characteristic lesions of arterio­

sclerosis.

The Role of the

Smooth Muscle Cell in Arteriosclerosis

Evidence that intimal smooth muscle, or a closely re­

lated cellular derivative, is involved in the formation of

connective tissue during atherosclerotic intimal thickening

of elastic and muscular artiers has been reported in both

human and experimentally induced atherosclerosis (Haust

et al., 1960; Geer and Haust, 1972; Ross and Glomset, 1973 )

It is not clear whether the modified smooth muscle found

14

in the intima orginates in the intima or migrates from the \

media. At birth most of ~he smooth muscle cells are located

in the medial layer of arteries and the i ntirna is compara-

tively thinner and contains only a few smooth muscle cells .

The internal elastic lamina represents a morphological bor-

der between the intima and the media. Fragmentation of the

elastic lamina has been shown to be associated with va~ious

forms of mechanical injury to the vasculature and would thus

permit the migration of medial smooth muscle into the intima

(Ross and Glomset, 1973). It has also been suggested that

the intact arterial endothelium normally acts as a barrier

to some substances present in the plasma which upon exposure

to vascular smooth muscle promote cell proliferation (Ross

and Glomset, 1973) . Smooth muscle cells have been reported

to accumulate in the intima at those sites in the arterial

vascular bed where endothelial permeability appears to be

increased (Helin et al., 1972) .

Benditt (1977) proposed that cells comprising athero-

sclerotic plaques have undergone mutational changes which

are analogous to the transformation process occurring in

benign tumor cells. This premise is based on the observa-

tion that cells within the plaques are monoclonal in origin

with respect to glucose-6-phosphate dehydrogenase i soenz yme

patterns (Benditt and Benditt, 1973 ) . These cells also

have morphological and functional features which are dif-

ferent from normal smooth. mu scle cells in the arterial wall

(Somlyo and Somlyo, 1968).

15

The Smooth Muscle Cell in Culture

Tissue culture, since it was introduced at the turn of

this century, has undergone several stages in its evolution.

In its present phase emphasis is placed on the analysis of

cellular interaction, cell differentiation, and cell func-

tion. Initial experiments have been conducted, establish-

ing the growth characteristics of normal rabbit smooth mus ­

cle cells in tissue culture (Ross, 1973; Doaud et al., 1964).

A pure population of smooth muscle cells can be iso­

lated from the tunica media of the aorta or large blood

vessels of most mammals. Therefore, if the adventitia and

intima are stripped off such ves sels, the remaining tunica

media is a source of pure smooth muscle cells for culture.

In culture, smooth muscle cells first acquire a fibroblast­

like appearance. During this stage many cells degenerate

but others proliferate. After a few weeks in culture the

daughter cells acquire the appearance of smooth muscle

cells (Ross, 1971). Their identity can be easily estab­

lished by electron microscopy because by then, they again

contain considerable amounts of myofilarnents in their cyt~­

plasm. The actornyosin in these cells can be demonstrated

by immunohistochemical methods (Knieriem et al., 1968 ) .

The similarity of these cells to cells in atherosclerotic

lesions make them a potentially important tool for athero­

genesis research.

16

Kokubu and Pollak (1961) observed that diseased arterial

tissue from cholesterol fed rabbits showed better cellular

outgrowth, after being placed in culture, than normal tissue.

In contras t to this Wexler and Thomas (1967) reported that

explants from arteriosclerotic breeder rats had a lower growth

curve than those from virgin rats . Fritz and co-workers

(1976) have been studying vascular smooth muscle for a num­

ber of years and have recently published information on pro­

tein synthesis and its relation to DNA synthesis in aortic

me dial explants . Their system consisted of free floating

explants of swine aorta media. Over an 18 day period protein

synthesis ( 14 c-1~ucine incorporation) paralleled DNA synthe­

sis ( 3H- thymidine incorporation). This is in agreement with

the generalization thatprotein turnover in slowly metaboliz ­

ing cells without cell division is minimal. However it was

pointed out by Osaki and Holtzer (1966) that for myogenesis

to occur, cells must withdraw from mitotic cycle for 5 to 8

hours of G1 before myosynthesis . Further studies of this na­

ture are definitely warranted to better establish relation­

ships between cell proliferation and the synthesis of

specialized proteins such as collagen and elastin, particu­

larly with regard to specific stimulatory factors which may

exist in serum and platelets .

Conspicuous accumulation of lipids by smooth muscle

cells existing in regions of arteriosclerotic lesions has

been mentioned earlier in this review. Cultured arterial

17

smooth muscle cells provide a useful system for studying the

differential effects of hyperlipemic serum and isolated lipo­

proteins (St . Clair and Leight, 1978; Bates and Wis sler,

1976). These studies suggested that differences in the serum

composition of lipoproteins resulting from genetic, dietary

or other environmental influences may play an important role

in determining the efficiency of delivery of cholesterol to

vascular smoot~ muscle cells and perhaps ultimately its

atherogenic potential.

Various studies, many of which have been cited in a re­

cent review by Ross and Vogel (1 978 1, support a hypothesis

that, in the absence of platelet function, the intimal smooth

muscle proliferative lesions of atherosclerosis can be pre­

vented. Therefore it is important to this discussion to

acknowledge that considerable current interest is being de­

voted to understanding not only the mode of action of plate­

let factors in cell culture, but its role in the stimulation

of a number of important in vivo proliferative cellular re­

sponses.

Cell Transformat.ion and the Collagen Phenotype

Medial smooth muscle cells are specialized mesenchymal

cells of the vasculature. The structural integrity of the

vessel wall depends upon the normal functioning of these

cells. It seems logical that a change in the differenti ­

ated state of vascular smooth muscle phenotype could lead

18

to the production of an abnormal matrix and to a loss of struc-

tural stability of such a tissue. Benditt (1973) has pro -

posed that cells comprising atherosclerotic plaques have

undergone mutational changes which are analogous to the

transformation process occurring in benign tumor cells. This

theory could be tested by determining if there is a change in

the heterogeneity of vascular collagen synthesized by smooth

muscle cells from arteriosclerotic vessels, since collagen

types are genetically prescribed.

A substantial amount of the information concerning cell

transformation and collagen synthesis has been obtained

from studying the transformation of chondroblasts into f ibro -

blasts. Recently it has been shown that when chick chondro-

cytes are grown in brom-d~oxyuridine iBUdR) for a few days,

collagen biosynthesis will switch from cartilage specific

type II collagen (chain composition [ci<'l (.II)] 3J to a mixture

2 of type I collagen lchain composition [o(l(I ) J2

and the

trimer (chain composition [ o<l(I)] 3 C1ayne et al., 1975 ) .

More recent investigations by that group showed that em-

bryonic chick chondrocyte possesses an inherent and latent

program of differentiation that involves "collagen switch-

ing" and by growing these cells in v itro it is possible to

potentiate this program (~ayne et al., 1976 ) . The altered

morphology of chick chondrocytes due to BUdR and due to

cellular senescence was very similar.

Another study reported that in suitable medium, chick

chondrocytes retain the morphology characteristic of

19

cartilage tissue and synthesize a matrix whose main constitu­

ent is chondroitin sulfate . ~hen chick embryo serum was added

to the culture medium the cells attained a fibroblastlike

morphology and stopped synthesizing chondroitin sulfate

(Marzullo and Lash, 1970). These authors proposed that under

suboptimal conditions, competition between two kinds of syn­

thesis for limiting levels of energy metabolites may favor

cell division over the production of tissue specific mole­

c~les. These tissue specific molecules could be considered

luxury molecules which are expendable and non- essential for

survival of a cell (Holtzer and Abbot, 1968) . This is con­

sistent with the findings of Mayne et al. (1 975 ) who ob­

served that the change in the phenotype of chick chondro­

c y tes grown in BUdR was characterized by an increase in DNA

synthesis, failure to accumulate extracellular matrix and

a "transformation" into motile cells that resemble fibro­

blasts.

It is therefore not surprising that somewhat similar

variability in the synthesis of collagen types has been re­

ported for medial smooth muscle cells in culture (Layman

and Titus, 1975; Barnes et al., 1976; Scott et al., 1977).

The difference s in the proportion of type I and III collagens

by these cells in culture may be a reflection of cell line

variability or differences in growth conditions employed.

Although it may be argued that such in vitro conditions

hardly resemble the in vivo situation (Fowler et al., 1977)

jt seems apparent that further studies if properly designed,

20

would be helpful in learning more about the regulation of

collagen heterogeneity and influences by the extracellular

matrix on gene expression. An understanding of those fac­

tors controlling collagen synthesis is basic to ou~ under­

standing of several disease processes including atherosclero­

sis.

Collagen Heterogeneity: Pathological Implications

Changes in the relative proportions of collagen types

within a tissue may occur in various disease states (Pope

et al . , 1975; Mcc ullagh and Belian, 1975; Seyer et al . , 1976;

Pent tinen et al . , 1975) . In one of these studies the im-

portance of type III collagen was indicqted by the suscepti­

bility of large arteries to rupture in patients with Ehler's

Danlos Syndrome (Type IV) who exhibited reduced levels of

type III collagen in these tissues (Pope et al., 1975). It

has been suggested that the arteriosclerotic plaques repre­

sent scar tissue, because a mixture of type I and II colla­

gens with a predominance of type I appears to be typical of

wounds .

During fetal development, the proportion of type I and

type III collagen molecules forming the dermis shifts from

a large proportion of type III to increasing amounts of

type I (Epstein, 1974 ) . Concomitant with this are changes

in mechanical properties and coarsening of the bundle fibers

(Pierard et al., 1976 ) . Such variations in bundle

21

organization observed in vivo occur in the same range of con­

centration as the most striking changes in bundle architec­

ture produced during in vitro assembly, i.e . from pure type

I to 30 % type III and 70 % type I. Work based on in vitro

reconstitution of type I and type JII collagen polymers,

demonstrated that the chemical structure of collagen molecules

determines the physic al structure of the bundles, type I

forming thick bundles of fibers and type III thin, isolated

fibers and mixtures of both type I and type III, bundles

with thickness varying with the respective c oncentrations of

the two type s of molecules (Lapiere and Pierard, 1977) .

Whatever the mechanism of the interaction of the colla­

gen types, it appears important during development and in

pathological conditions. It would explain the resistance to

· stretching observed for thick bundles of type I collagen

in tendon in contrast to the laxity and the distensibility

of the blood vessel wall, which contains a mixture of type

I and type III collagens (Lapiere and Pierard, 1977) . Also,

Hughes et al. (1976) has reported that in vitro platelets

are known to adhere better to type III collagen than to

type I. However, there are arguments regarding the correla ­

tion and interpretation of in vitro experiments involving

type-specific-collagen : platelet interactions.

It has not been established if type I and t ype III

collagens form two populations of fibers or one population

composed of type I and type III molecules. In all the

22

conditions mentioned above the altered collagen composition

and the increased proportion of type I are accompanied by

the presence of coarser bundles and impaired mechanical func-

tions. Although type III ()(-chains have the distinction of

containing two, half-cysteine residues, there are no contrast­

ing features regarding the elemental composition of each

type which could better explain the individual and composite

architectural properties of collagen type I and III.

EXPERIMENTAL

Animals

Male albino New Zealand rabbits weighing 3 to 4 pounds

were obtained from Glocester Rabbitry (Glocester, Rhode

Island) at least one week prior to the start of the experi-

ments. The animals were housed in pairs in a roon main-

tained at an ambient temperature of 24°-27°C., with alter-

nating 12 hour light/dark cycles. All animals were offered

a commercial laboratory chow and water ad libitum.

Materials

Analytical grade reagents were used throughout the in­

vestigation. The following drugs were purchased: L-epine­

phrine bitartrate and L-thyroxine (free base) (Sigma Chemi­

cal Company, St. Louis) and 3-aminopropionitrile fumarate

(Aldrich Chemical Company, Milwaukee) . The radioactive

23

amino acid 2,3- 3H-L praline (spe ~ ific activity approximately

20 Ci/mMo.) and methyl- 3H-thymidine (52.l Ci/mMol) were

purchased from New England Nuclear Corporation (Boston) .

Chromatographically purified clostridial collagenase was

obtained from Advanced Biofractures, Inc . (Lynbrook, New

York ) and pepsin (2500 U/mg) from Worthington (Freehold,

N.J.). The purity of the collagenase was confirmed by its

24

14 inability to degrade C-tryptophan labeled E. coli protein.

Type I and Type III collagen standards were prepared by

Ronald Goldberg (U .R. I.) by differential salt precipitation

of acetic acid solubilized newborn calf skin.

Electrophoretic grade acrylamide and N,N' - methylene-bis -

acrylamide, N,N,N' ,N'-tetramethylethylenediamine and dodecyl

sodium sulfate were purchased from Eastman Kodak Co.

(Rochester, N.Y.). Carboxyrnethylcellulose CM52 (Whatman,

Kent, England) was used for chromatographic analysis.

Biologicals for the cell culture and organ culture sys-

terns were obtained from Gibco (Grand Island, N.Y.) and in-

eluded: Dulbecco's Modified Eagle Medium (with glutamine),

fetal calf serum (heat inactivated), amphotericin-B (250

mcg/ml), Penicillin-Streptomycin (10,000 U/ml-10,000 mcg/ml)

and pancreatin (2.5%) .

Epinephrine-Thyroxine Induced Arteriosclerosis

Gross aortic plaques were induced in male New Zealand

rabbits by daily injections of epinephrine and thyroxine

for 15 days (Fuller et al., 1976) . Epinephrine (0 .025

mg/kg for the first 5 days; 0.05 mg/kg thereafter) was

injected via the marginal ear view with an infusion pump

(Harvard Apparatus Company Inc., Model 940, Dover, Massa-

chusetts). Thyroxine (0.05 mg/ kg) freshly prepared was

injected intraperitoneally. Animals were killed by cervical

dislocation 24 hours after the last injection . The thoracic

25

aorta was removed under aseptic conditions and rinsed with

cell culture medium. The adventitia and endothelium from

plaqued regions and from analogous regions of untreated con­

trol rabbits was removed. The remaining intimal-medial

tissue was cut into 1 rnm2 pieces which served as the source

of aorta smooth muscle for organ culture and for growing

cultures of smooth muscle cells (Fedoroff, 1973).

Organ Culture System

Approximately 50 mg aliquots of freshly dissected medi­

al smooth muscle from each animal were placed individually

in 2 ml of Dulbecco's Modified Eagle Medium (DMEM) contain­

ing 100 ug/ml of 3-aminopropionitrile (BAPN) and 400 ug/ml

of ascorbate. After a 2 hour preincubation at 37°C in

5%C02-95% air, 100 uCi of 2,3- 3H-L praline was added to each

sample . Incubation was resumed for another 70 hours, after

which the samples were transferred to a freezer for storage.

Labelled collagen was extracted by first homogenizing the

smooth muscle tissue in its 2 ml incubation media using a

conical ground-glass homogenizer. A 400 ul portion of this

homogenate was taken for DNA determination according to the

method of Burton (1956). The remainder of the sample was

dialyzed against 0.5M acetic acid and then treated with

pepsin (100 ug/ml ) at 4°C for 12 hours. After limited

proteolysis of noncollagenous protein, tissue debris was

removed by centrifugation at 30,000 rpm for 20 minutes.

26

The supernate was then dialyzed for 12 hours, against 0.05M

phosphate buffer (pH 7.2) and the volume adjusted to 1 . 6 ml.

Aliquots of these sam?les could be combined with electro­

phoresis buffer, heat denatured (56°C x 20 minutes) and the

labelled alpha chains separated on 5% polyacrylamide gels

according to the method of Neville (1971).

Culturing of Smooth Muscle Cells

A portion of the medial tissue was also used as explant

culture to derive colonies of smooth muscle cells for each

of the experimental animals. The 60mm Falcon petri di shes,

each containing approximatel~ 5 to 10 self-adhering explants

from a specific animal, were flooded with 10 ml of DMEM.

This medium contained 20% fetal bovine serum, penicillin

(100 U/ml), streptomycin (100 ug/mll and amphotericin B

t2.5 ug/ml). The explant cultures were incubated at 37°C

in an atmosphere of 5%co2-95 % Air. After the appearance of

sufficient marginal outgrowth from the explants (about 18

days )_ the media was removed and the cul tu res rinsed with

phosphate buffered saline (pH 7.0}. The cells were de­

tached by a 15 minute incubation at 37°C in 0.25 % Pancreatin

(.N. F. )_ solution (pH 7.4)_ and transferred to new 60mm petri

dishes with fresh medium containing serum. This pancreatin

solution, used in the manner described by Merchant et al.

(1969 ) , was more effective in removing cells from the

27

culture flasks than 0.25% trypsin solution. Subculturing was

performed at least once a week thereafter, or whenever a dish

reached confluency. By seeding successively larger dishes a

population of 5 X 610 cells/ 75 cm2 was reached at about the

4th passage. At the end of the 4th passage fresh medium

(DMEM) was added which contained only 10% fetal bovine serum

plus 100 ug/ml BAPN, 400 ug/ml ascorbate and 2 uCi/ml 2,3-

3H-L praline. This system was incubated under the conditions

described above, £or 12 hours. After incubation the media

was removed and stored frozen. The cell layer, for measure­

ment of total DNA, was removed with 0.1 % Pancreatin solution

(Gibco) and the cell pellet stored in the freezer.

The medium from each flask was treated with 50 % anunonium

sulfate to precipitate protein. The precipitate was redis­

solved in 1 ml of O.SM acetic acid containing 20 mcg of

pepsin and dialyzed in the same manner for 12 hours at 4°C.

The pepsin was inactivated by redialyzing against O.OSM

phosphate buffer (pH 7.2). After dialysis each sample was

adjusted to 2.0 ml with O.OSM phosphate buffer (pH 7 .2 ) and

stored in the freezer.

Thyrnidine Incorporation by Explant

Cultures of Vascular Smooth Muscle Cells

Labelled thyrnidine incorporation was measured at day 18

in explant cultures prepared as described above. Each 60 mm.

dish contained 5 explants (1 mm2) and all five explants

28

were taken from the same animal. Five replicate dishes were

prepared for each animal. At the 17th day of incubation

under the previously described conditions, the medium was

replaced. On the 18th day 0 . 01 uCi of 3H- thymidine was

added to each petri dish andincubated for 8 hours. The medium

was rerncved and the cells detached with 1 ml of 0.25 % pan-

creatin solution. The cell suspensions were precipitated

with ice-cold 5% trichloracetic acid (7CA) and the pellet

washed 3 times with 2 ml volumes 5% TCA. The washed pellets

were sonicated in 1 ml of 0.5N perchloric acid. The radio­

activity was determined in 200 ul. aliquots of the perchloric

acid in solution Hydrornix® , by liquid scint illation spec­

trometry .

Polyacrylamide Gel Electrophoresis

The 5 % polyacrylamide gels and electrophoresis buffers

used in this part of the study were prepared by the methods

described by Neville (1971) . To a 50 ul. aliquot of each

sample, described above, 50 ul. of sample preparation buf­

fer was added . This sample preparation buffer consisted of

.02M phosphate buffer (pH 7 . 2), 2M urea, 0.2% sodium

dodecyl sulfate, 10% sucrose and contained 1 mg/ml of a 1:1

mixture of purified type I and type III calf skin collagen.

Each sample wa s prepared in duplicate; one of which was

made 2% with mercaptoethanol to reduce the disulfide link­

ages in type III collagen. All samples were then heat

29

denatured for 20 minutes in a 56°C water bath. Aliquots of

the reduced and unreduced (100 ul. each) were loaded onto

separate gels and electrophoresed at 2 mAmps per gel for ap­

proximately 2.5 hours. The gels were then stained in 0.5M

acetic acid containing 0.25% Coomassie blue. The regions

corresponding to the collagen alpha chains were cut out and

dissolved in 30% hydrogen peroxide . The radioactivity in

each band was determined by adding liquid scintillation

cocktail (Hydromix by Yorktown) and counting in a Packard

spectrometer.

Carboxymethylcellulose Chromatography

The constituent alpha alpha chains of collagen were sep­

arated by CM-cellulose chromatography essentially as de­

scribed by Miller et al., 1971. The column (1 .5 x 8 cm ) was

equilibrated with the starting buffer 0.05M sodium acetate

(pH 4.8) and after application of sample, eluted with a

linear gradient of sodium chloride from 0 - O.lM in starting

buffer, over a total volume of 400 ml. All CM-cellulose

chromatography was carried out at 45°C.

Four individual samples from each experimental group

had to be pooled to obtain sufficient radioactive material

( ~ 200,000 cpm ) for separation and quantitation of collagen

types by CM-cellulose chromatography. After dialyzing

these pooled samples against starting buffer, urea (0 .5

g/ml ) and 2-rnercaptoethanol (2 %) were added. Each sample

30

(cpm) in the ~2 band and the ~l band (with and without

reduction by rnercaptoethanol) were used for the calculation

of type I and type III collagen. Type I was represented

by the cpm in o<l (without reduction) p.lus cpm in ~2.

The radioactivity in ~2 was not affected by reduction.

These values were multiplied by the appropriate dilution

factor and expressed per ug. of DNA in the total organ

culture or cell culture system.

The radioactivity in 1 ml aliquots of each fraction was

plotted and the peaks representing the labelled collagen

peptides ( o<l [I], o<l [III] ,o<2) were quantitated by measur-

ing the area under each peak with an electronic planimeter

(Numonics Corp., Landsdale, PA.). Type I was quantitated

from the combined area of the peaks for ~l and ~2 chains.

Type III was represented by the area of the ~l(III) peak.

Although these values could not be accurately expressed

per ug of DNA, they were expressed as ratios of type III:

type I and compared with the electrophoretic data similar-

ly expressed.

Statistical Methods

a) Mean: x = ~ ~ ~. l

b)

where n = sample size

x. = some of values for each sample l

Standard deviation:

2 where s = 1 n-1

s=~

~ (xi-x) 2

31

from che combined area of the peaks for o<l and ~2 chains.

Type III was represented by the area of the o<l(III) peak.

Although t~ese values could not be ~ccurately expressed per

ug of DNA, they were expressed as ratios of type III: type I

and compared with the electrophoretic data similarly ex-

pressed.

Statistical Methods

= 1 a) Mean : x n

b)

where n = sample size

x. = some of values for each sample l.

Standard deviation:

2 where s =

1 n-1

- 2 (X·-X)

l.

c ) Standard error of the mean: S~ = s/~

d) Student "t" test: '\{cN1-1) s~ + (N

2-1) s~

Level of significance was set at p < .05.

32

e) Chi squar9 te st (for two independent samples)

r k 2 x2 ~ ~ (0 - E )

= ij ij i=l i=l "t"

~'' l. J

where 0 .. = observed number of cases (explants categor­l. J

ized in the ith row of jth column.

E ij

= number of cases (explants) expected under H 0

to be categorized in ith row of jth coluni.n .

r

~ directs one to sum over all (r) rows

i=l j =l

2 and all (k) columns. The values of 'X yielded by the above

formula are distributed as chi square with cf = (r-1 ) (k-1),

where r = the number of rows and k = the number of

columns in the contingency table. To find the expected

frequency of each cell (Eij), multiply the two marginal

totals common to a particular cell, and then divide this

product by the total number of cases (explants) N.

RESULTS

This investigation used an established method of epine­

~hrine infusion with thyroxine administration to produce

~acroscopic fibrous lesions in rabbit aorta (Fuller and

Langner, 1970 ; Mickulicich and Oester, 1970; Lorenzen, 1962)

Histopathological changes in the aorta included among other

?rominent features (Fuller et al., 1976): pronounced inti­

mal thickening and the proliferation of a specialized cell

type in the media. Vascular tissue for this investigation

was obtasined from the medial region of the aorta which is

composed predominantly of smooth muscle cells (Ross and

Glomsett, 1973; Bierring and Kobayasi, 1963) . There are no

fibroblasts present in the media of mammalian arteries in

contrast to the arteries of other species such as birds

(French, 1966).

This investigation was conducted to examine the rela­

tionship between the proliferation of medial smooth muscle

cells in culture and changes in the t ype specific synthe­

sis of collagen related to atherogenesis. Factors which

influence this interrelationship appear to play a role in

the development of the fibrous vascular plaque . This line

of investigation would identify fundamental changes in the

function of cells derived from arteriosclerotic smooth

muscle since the ratio of collagen type synthesized is con­

trolled by gene expression.

33

Vascular Smooth Muscle Cell Proliferation

Medial explants (1 rnm 2 ) from corresponding regions of

the thoracic aorta of normal and arteriosclerotic rabbits

(same age) were incubated under the established conditions

described in the Methods section . After 12 days of incu­

bation the expla~ts were observed and their growth was

graded according to their appearance under the Microscope

at a magnification of lOOX. P"ive explants were attached

in each 60 mm petri dish anct each explant in the dish was

qraded. Five dishes were seeded frcm each aorta for a total

of 25 explans per animal . Explants bearing a confluent

mat"-ix of smooth muscle cells at day 12 were designated:

GOOD GROWTH. Those explants giving rise to fewer smooth

muscle cells and lacking confluent orientation were desig­

nated as SOME GROWTH. NO GROWTH designated those explants

with no evidence of cells migrating from the explant.

Representative photomicrographs of these categories are

shown in Figure 1. The tabulated results of this evalua­

tion are shown on Table 1 and incicate the mean percentages

of explants for each growth category. In the control group

14% of the explants showed GOOD growth of smooth muscles

cells; 58% SOME grcwth and 28 % NO growth. In the arterio­

sclerotic group 60 % of the explants showed GOOD growth;

34

7% SOME growth and 37% NO growth. Experiment 2, performed

independently from Experiment 1, used a sufficient number of

FIGURE 1

PERIPHERAL

GOOD GROWTH

A Confluent Matrix of Cells (lOOX)

SGlME GROWTH

Significantly fewer cells, lacking confluent orientation (lOOX).

35

animals in each group to permit statistical analysis by the

Student "t" test . In Experiment 2 a Chi Square test for

nonparametric data was used to test for significant differ­

ence between the control and arteriosclerotic groups regard­

ing the absolute number of explants assigned to their appro­

priate growth categories (e.g . , GOOD, SOME, and NO growth) .

This analysis also indicated significant differences between

control and arteriosclerotic in the GOOD and NO growth cate­

gories . There was no noticeable difference in morphology

of the cells in each group .

Measurement of smooth muscle cell proliferation from

the medial explants was determined for each petri dish on

the 18th day of incubation by incorporation of 3H- thymidine

into TCA precipitable protein. The early indication of

smooth muscle cell hyperplasia in arteriosclerotic tissue

is further substantiated by the 2-fold increase in 3H­

thymidine incorporation (CPM/ ug DNA) during an 8-hour label­

ing period. The increase in m1A due to new cell growth was

below the level of sensitivity of the colorimetric assay

(Burton, 1956) and the DNA value 4.6 ug reflects, for the

most part, the combined DNA of the five tissue explants in

each culture dish.

Collagen Heterogeneity in Primary Cultures of

Rabbit Medial Smooth Muscle Cells

Thoracic medial smooth muscle from normal and arterio­

sclerotic rabbits was incubated with 3H-L proline in an

36

37

TABLE 1 a

ESTIMATION OF NEW CELL GROWTH

FROM MEDIAL EXPLANTSb

Experiment 1

c Growth Control-(2 animals) Arteriosclerosis-(3 animals)

Good

Some

None

14%

5 8%

28%

d Experiment 2

60%

7%

37%

Growthc Control - (3 animals) Arteriosclerotic-(4 animals)

Good

Some

None

a

14% + 7

48% + 8

30% + 10

56% + 9e

32% + 7

Observed under light microscope (lOOX) at day 12 of incuba ­tion (37°c, 5% co 2/95% Air).

bThoracic aorta medial smooth muscle (1 mrn 2 ) 25 explants per rabbit and 5 explants per 60 mm dish.

cGrade of growth: Good = confluent matrix of outgrowing cells Some = presence of growth but no confluent matrix of cells; None = no outgrowth detectable.

d Experiment 2 conducted inde~endently from Experiment 1.

e Values are mean+ S.E. and significantly different from

control (p <. • 0 5 Student "t '' test) .

p 1,1 ~1.2

FIGURE 2

A 8

c< l(I )

<X2

38

~gration of Type I and Type III Collagen Standards on ?oly­acrylamide gels Before (A) and After Reduction With 2% ~1ercapto­e t hanol (B). The slower mobility of Ol\l (II I ) is apparent.

FIGURE 3

EFFECT OF MERCAPTOETHANOL AND BACTERIAL COLLAGENASE TREATMENT

ON THE ELECTROPHORETIC MIGR.A.TION OF 3H- PROLINE LABELLED COLLAGEN

SYNTHESIZED IN CULTURE BY RABBIT VASCULAR SMOOTH MUSCLE

6000 (A\

2000

~ w 7

u ....) (()

E 6000 ( j)}

E ...,. er w 0...

w 2000 z _j

0 tr 1 a..

I

I ff)

E Q.. f500 ( ( ) ()

1000

500

5 7

cm MIGRATED FROM TOP OF GEL

Legend for Figure 3 on page 4~ .

40

FIGURB 3. Effect of 2-Mercaptoethanol and Bacte~ial Collagenase Treatment of the Electrophoretic Migration of 3H-Proline Lablelled Collagen Synthesized in Culture by Rabbit Vascular Smooth Muscle.

41

Sample preparation and electrophoresis conditions described in the Methods. Each point represents the radioactivity in 4 nun. slices of the gel.

Panel A: Panel B:

Panel C:

Unreduced sample of extracted collagen. Identical sample reduced with 2%

mercaptoethanol. Identical sample which was previously

incubated with purified bacterial collagenase (protease-free ) .

a.

b. c. d.

CONTROL

e AR'I'ERIOSCLEROTIC

TABLE 2

PROLIFERATION OF SMOOTH MUSCLE CELLS FROM

a EXPLANTS OF RABBIT THORACIC MEDIAL 'I'ISSUE

Nb

(10)

(12)

3 h .d. c cpm H-t yrni ine

1661 + 571

3230 + 347

d Total DNA (ug)

4.6 + 0.2

4.6 + 0.2

cpm/ug D~A

364 + 125

f 708 + 76

Approximately 25 explans from each animal. Five 1 mm2

explants per dish (60 X 15 mm.) At 18th day of incubation as described in methods.

Number of dishes (60 X 15 rrun) in each group. All Cultures labeled with 3H-thymidine (10 uCi) for 12 hours. Per dish (explants plus cell layer) by the method of Burton, 1 956.

e. Arteriosclerosis induced by epinephrine and thyroxin administration.

f. Mean~ S.E.; significantly different from control (p <.05) by student's "t" test.

""' I\.)

43

o(-region due to heat denaturation in the presence of 2%

2-mercaptoethanol . Panel (C) in Figure 3 shows loss in radio ­

activity due to prior incub~tion of the collagen preparation

with purified clostridial collagenas e (Peterkofsky &

Diegelnann, 1971) .

The increased radioactivity in the Q(.- lregion after re­

duction with rnercaptoethanol indicates that control rabbit

vascular smooth muscle synthesized 50 % type I collagen and

50% type III collagen (Table 3) . Smooth muscle tissue from

plaqued regions in the arteriosclerotic groups synthesized

63% gype I collagen and only 37% type III collagen. In­

creased synthesis of type I and III collagen in the arterio­

sclerotic group was significantly different from synthesis

in the control group at p ~ . 05 (Student "t" test) . This

could alternatively be expressed as a statistically sig­

nificant change in the type I/type III ratio: 1.0 for the

control group and 1.7 for the arteriosclerotic group. The

s ynthesis of total radioactive t ype I and type III collagen,

per ug of DNA, extracted from the primary cultures increased

approximately two-fold in the arteriosclerotic group ccm­

pared to control .

Collagen Heteroqeneity and Vascular

Smooth Muscle Cells in Culture

Thoracic medial smooth muscle explants from normal and

arteriosclerotic rabbits were placed in culture as described

TABLE 3

COLLAGEN SYNTHESIS IN ORGAN CULTURE

BY RABBIT MEDIAL SMOOTH MUSCLE

CONTROL (N=l4) ARTERIOSCLERO'rIC (N=l 3)

Type I cpm/ug DNA 28 3 + 31 743 + 84*

Type III cpm/ug DNA 254 + 25 437 + 46*

Total 537 + 37 1180 + 96*

Aorta medial smooth muscle was incubated for 72 hours at 37°c in 2 ml of DMEM con­taining 2, 3- 3H-proline, BA.PN, and ascorbate. Collagen types separated on poly­acrylamide ge ls and quantitated radiometrically .

Values are the mean + S.E.

*Significantly different from control (p <. .05) Student "t" test.

.i::.

.i::.

45

in the Methods and the previous experiment. Daughter cells

from these explants were propagated in DMEM containing 20%

fetal calf serum and antibiotics . These cells were subcul-

tured approximately weekly in~o successively larger flasks

6 2 up to a confluent population of 5 X lO cells per 75 cm .

This stage of growth represented 4 passages . At the end

of this passage the cells were incubated for 24 hours in

medium containing 3H- proline (2 uCi / ml), BAPN (100 ug/ ml)

and ascorbate (400 ug/ml) . Labelled collagen was extracted

from the culture medium and DNA determined in the cell

layer. Collagen types I and III were quantitated on gels

according to the method previously described . Table 4

shows the relative and total amounts of type I and type III

collagen synthesized by smooth muscle cells (SMC) at the

end of their 4th passage . Daughter cells from normal aorta

synthes ized 61% type I and 39% type III at this passage

while daughter cells from arteriosclerotic medial tissue

synthesized 78% type I and only 22% type III collagen . Com-

bined values for type I plus type III indicated that total

collagen synthesis, although somewhat lower for the arterio-

sclerotic group (11, 174 ~ 1338 cprn/ug DNA versus 13,230

~ 1150 cpm/ug DNA) was not significantly different from

controls at the p <.OS level.

Collagen Synthesis and Age in Culture

Collagen · t ype I and type III was measured in SMC cul-

tures at the 10th passage . Figure 4 i_lustrates relationship

'l'ABLE 4

COLLAGEN SYNTHESIS IN CULTURE

BY RABBIT MEDIAL SMOO'rlf MUSCLE CELLS

CONTROL (N=l2) ARTERIOSCLEROTIC (N=l5)

Type I cpm/ug DNA 7963 + 499 8750 + 949

Type III cpm/ug DNA 5049 + 823 2424 + 486*

'rot al

Confluent for 24 hours calf serum. metrically.

13230 + 1150 11174 + 1338

6 2 cultures ~5 X 10 cells/75 cm ) at the fourth passage. Incubated with 2,3- II-praline in the presence of BAPN ascorbate and 10% fetal Collagen types separated on polyacrylamide gels and quantitated radio­Values are the mean + S.E.

*Significantly different from control (p < .05) Stude nt " t" test .

"" °'

FIGURE 4

EFFECT OF CULTURE PASSAGE NUMBER ON THE S~THESIS OF TYPE I AND

TYPE III COLL.A.GENS BY AORTA MEDIAL S~fOOTH XUSCLE CELLS FROM

NORMAL AND ARTERIOSCLEROTIC RABBITS

. LLJ . Cl)

..p I

~ 0

~ l.LJ a.. >-f-

~ 0

100

c

~o

10

TYPE I c:::::J TYPE m ~ C :a CONTROL

A= ARTERIOSCLEROTIC

A

A c

0 4

PASSAGE NUMBER

c .. A

• 10

ALTERNATIVE REPRESENTATION OF TYPE III SYNTHESIS

100 CONTROL -ARTERIOSCL EROTIC

~o

.... "-C- ... -- --

0 --·-.-0 4 10

PASSAGE NUMBER

47

48

between age in culture and the relative synthesis of type I

and type III collagen. The cells from arteriosclerotic tis­

sue make a larger proportion of type I (vs type III) com­

pared to control at all passage levels. By the tenth pass­

age type III synthesis represents no more than 10% of the

collagen synthesized by cells grown from normal or arterio­

sclerotic tissue. It was also apparent that by the tenth

passage the cells in both groups were layi~g down less matrix

compared to earlier passages which allows these cells to

be more easily dissociated (enzymatic) upon subculturing,

and to grow in confluent monolayers. In contrast, cells

at early passages grow on top of one another, resembling

hills and valleys, before reaching confluency.

Collagen Heterogeneity by CMC-Chromatography

The electrophroeticseparation of labelled collagen

types was developed for this study in order to permit ana­

l ysis of individual cultures in sufficient numbers for

statistical comparison . In order to validate and confirm

the data from this gel-electrophoresis system, pools w~re

prepared from the primary organ culture system and also

from the cell culture system to obtain sufficient radio­

active material to analy ze collagen t ypes on carboxymethyl­

cellulose as described in the ~ethods.

After loading the reduced col_agen extracts, the

collagen was eluted from the CMC with 400 ml of sodium

acetate buffer (0.06M, pH 4.8) over a continuous NaCl gra ­

dient (0.0 to O.lM) and 10 ml. fractions were collected.

The elution pattern is reflected by the radioactivity in

aliquots from each fraction (Figure 5). Moving from left

to right on the chromatogram in Figure 5 the three peaks

after the gradient correspond to ~l(I); ~l (III) ; and ~2.

In Figure 5 (Organ Culture) the ~l(III ) peak is high in

relation to ~l (I) for control tissue (upper panel) and is

relatively low in relation to ~l(I) for the arteriosclero­

tic tissue group shown in the lower panel. As shown in

Figure 6, there is a similar decrease in relative amount

49

of radioactivity in the ~l (III) peak in the arteriosclerotic

group (lower panel) compared to the control group shown .in

the top panel. The ratio of type III: type I collagen was

calculated from the total radioactivity found in each peak

and is compared with the values obtained by gel electro ­

phoresis (Table 5) . For the organ culture system the ra­

tios of collagen type III:I determined by either method were

nearly identical. For the cell culture system both methods

indicated an increase in type I collagen, relative to

type III, for the arteriosclerotic group; however, the ra­

tios of III:I calculated by CMC-chrornatography were general ­

ly higher than those obtained by the electrophoretic method

(Table 5) . A significant difference in the results by the

two analytical methods appeared only in the control cell

culture group . This difference can be accounted for by the

FIGURE 5

CMG-Chromatography of Labelled Collagen Synthesized In

Organ Culture By Aorta :iedial Smooth Nuscle From Control

And Arteriosclerotic Rabbits.

400 1-·1 GR. o(l(I) o<l (III) o<2 I

i t ,i I I l I

300 I I I I I I

200 I I

E I I

...... I \ I \ __ ~ E 100 I

Q. I Control I u I

0 0 ~10 20 w 400 ,-, i i 2 t I

_J : I l 0 300 et: I I

a. I I

I 200 I I I :c I

If) I 100 I I Arteriosclerotic , ___ -

0 0 10 20

FRACTION NUMBER

Top Panel: Control Gro:p. Bot ~om Pa~el: Arterioscleroltic Group. Collagen extrac:s from nor:nal and arteriosclero tic groups were ?re?ared and applied to the column as described in the M~thods. The bound collagen was eluted with a contin­uous sodi~~ chloride gradient (O - 0.1 ~).The eluate was collacted ~n lO ~~ . fractions and the radioactivity in 1 ml. aliquots 0£ each ccunted. T~e pos ition of elution of collagen ~l(I), o<l(III ) and OC:2 chai~s was :stablisheci oy chromatography of calf skin collagen 3tanJ~rds.

50

E

' E

FIGURE 6

CMC-Chromatography of Labelled Collagen Synthesized by Cultured Smooth Muscle Cells Derived From Aorta ~edia of Control and Arteriosclerotic Rabbits.

3000 ,--1 GR. oCl(I) e< l (III) 0<2

I l

i I I l i I l

I I I

I I 2000 I I I l I I I

1000 I I I ,

c. I I (,) I I Control --

0 I w z 3000 I\ 0 20

...J I \

l t l 0 I I

a:: ' \ a.. I I I I I

:t: 2000 I')

I I

' I I

\ 1000 \

0 20

FRACTION NUMBER

.Top Panel: Control Group. Bottom Panel: Arteriosclerotic Group. Collagen extracts were prepared and applied to the column as described in the Methods. The bound collagen was eluted with a continuous sodium ·chloride gradient (0 - O.D1). The eluate was collected in 10 ml. fractions and t he radioactivity in 1 ml. aliquots of each counted. The position of elution of collagen o( 1 (I) , o( l (III ) and o( 2 chains was established by chromatography of calf skin ccllagen standarcis.

51

a

b

TABLE 5

COMPARISON OF ELECT!\OPHORETIC AND CHROMATOGRAPHIC RESULTS

FOR THE SYNTHESIS OF TYPE I AND TYPE III COLLAGENS

group Gel Electrophoresisa

CONTROL-Organ Culture 1 le

ARTERIOSCLEROTIC-Organ Culture 1 1.7

CONTROL-Cell Culture 1 1. 5

ARTERIOSCLEROTIC-Cell Culture 1 3.6

Mean values from Tables 3 and 4 (cpm/ug DNA) .

b CMC Chromatography

1 1

1 1. 9

1 2.5

1 4. 5

Ratios computed from areas under the peaks of elution profile for labelled collageno us proteins.

cRatios represent amount of type III:I collagen.

IJ1 N

unavoidable variability in the individual ratios - for the

samples that were pooled for CMC-chromatography . Except

for the above-mentioned group the variability between the

two methods was no more than 10%.

53

DISCUSSION

Intravenous injections of epinephrine in the rabbit

produces arteriosclerotic lesions, which are aggravated by

the simultaneous administration of L-thyroxine. The major

alterations observed in rabbit arteriosclerosis induced by

epinephrine plus thyroxine occurs in the smooth muscle cells

and the elastic fibers of the aorta, similar to the lesions

seen in arteriosclerosis of man (Kobayasi, 1969; Daoud et

al., 1964). Histological examination reveals structural

changes in the smooth muscle cells such as cyctic protru­

sions of the sarcoplasm, vacuole forma~ion and condensation

of myofilaments. These changes are accompanied by frag­

mentation degeneration of the elastic fibers. It ha s been

suggested that factors, associated with various forms of

vascular injury, influence the fundamental structure and

function of the medial smooth muscle (Ross and Glomset,

1973). It has been proposed that medial smooth cells, re­

sponding to intimal injury, migrate into the intirna and

begin to proliferate establi s hing a colony rese~bling a be­

nign neoplasm which synthesize a generous amol1.nt of colla ­

gen (Benditt, 1977). McCullagh and Belian (1975) analyzed

pe?sin extracted collagen from a pooled sample of intimal

pla.ques from 8 atherosclerotic ~u.TT\an aortas. They reported

5 :

a marked increase in the ratio of type I to type III collagen,

55

compared to that in the nonarteriosclerotic aortic media .

In their procedure, less than 50% of hlli~an aorta collagen

was recovered in the extracts and subsequently characterized.

No studies have reported the relative rate of synthesis of

type I and III collagen in diseased vascular tissue.

The data described in this investigation establishes

that a significantly greater population of smooth muscle

cells migrate out of the medial explants dissected from tis­

sue subjected to an atherogenic condition compared to

medial explants from the nonarteriosclerotic group. This

enhanced outgrowth from tissue explants supports the cur­

rent hypothesis that in atherogenesis modified smooth muscle

cells in the medial layer migrate from the media, through

the internal elastic lamella and into the intimal region.

Conditioned media from rapidly growing medial explants

when transferred to slower growing explants did not stimu­

late migration of cells from slower growing explants. This

observation would rule out the possibility that the rapid­

ly proliferating and migrating modified smooth muscle cells

liberate soluble growth promoting factors. However, there

may be a higher concentration of platelet factors, insulin

or lipoproteins, due to increased vascular permeability, in

the explants of arteriosclerotic tissue which could enhance

SMC outgrowth (Ross and Glomset, 1973; Friedman et al., 1976)

Also the accumulation of collagen, elastin and mucopol y ­

saccharides in arterioscler0tic tissue may present an extra­

cellular matrix more supportive of cellular outgrowth and

migration.

Another explanation for the more rapid outgrowth could

be based on decreased protein degradation and higher meta-

bolic activity in the tissue explant. The first evidence

that decreased protein degradation may contribute to rapid

growth of mammalian tissues came from studies of work in­

duced hypertrophy of skeletal muscles (Goldberg et al.,

1975). Similar reports exist in the literature correlating

necreased proteolysis with rapid compensatory growth in

other tissues (Scornik, 1972). Current understanding of

the relationship between DNA synthesis and protein synthe­

sis leads to the conclusion that, over an extended period

of time, DNA s ynthesi s must be dependent on protein syn­

thesis. However, it is known that the high dose of thyroid

hormone used for the experimental model in this study pro ­

duces generalized weight loss and muscle atrophy. Under

such conditions total protein degradation should exceed

total protein synthesis. Therefore, rapid outgrowth of

medial explants from arteriosclerotic vessels appears to

be the result of some phenomenon which specifically stim­

ulates a latent growth property in vascular smooth muscle

cells.

As discussed above, enhanced cell growth in the ar­

teriosclerotic group was observed at the 12th day of i~cu­

bation. At the 18th day in culture 3H-thymidine incorpor­

ation into DNA indicated that smooth muscle cells in the

arteriosclerotic group were still proliferating faster than

56

57

SMC from control explants. Stimulatory factors present in

the tissue explant could have been responsible for promot­

ing early outgrowth, but it is unlikely that they are still

influencing new DNA synthesis at the 18th day in culture in

the presence of fresh media. This data is also supported

by the consistent finding that it took comparatively long­

er to propagate smooth muscle cells in the control group to

their fourth passage . Increased mitotic activity is a

common characteristic of transformed or the differentiated

cells. The ability of SMC from arteriosclerotic tissue to

adapt to and proliferate more rapidly in vitro is suggestive

that they have undergone fundamental changes and could be

considered analogous to those cells comprising the benign

smooth muscle tu~or described by Moss and Benditt (1975)

and .Rounds et al. (1976).

Morphologically , S~C grown from normal tissue and ar­

teriosclerotic tissue were indistinguishable and they both

exhibited t ypical features such as abundant myofilaments

and surface vesicles. These observations agree with those

of Ross (1971) who has used identical culture methods.

Further characterization of these cells was not within the

scope of this study , but has been explored by other in­

vestigators (Mauger et al., 1975; Ki~es and Brandt, 1976)

who obtained SMC by the same technique . However, it was

observed in this study that SMC f rom normal tissue enter

senescence sooner than SMC from a rteriosclerotic tissue.

58

Senescence was accompanied by p ronounced vacuolization, loss

of cellular definition, and loss of proliferative activity.

A portion (50 mg) of freshly dissected medial smooth

muscle was placed in an organ culture s y stem as described

in the Methods. Preliminary experiments indicated that 50

uCi/ml of 2,3- 3H-L praline and a 72-hour incubation were

necessary to obtain adequate quantities of newly s y nthe­

sized soluble collagen of high specific activity for reso­

lution on polyacrylamide gels. The use of BAPN prevents

crosslinking of collagen molecules and assu"-es complete ex­

traction of the labelled collagen. These primary explant

cultures maintain viability for prolonged periods based on

a report by Fritz et al. (1975) that protein and DNA syn­

thetic activity persist for up to 9 days in aortic medial

explants that were Maintained under similar conditions.

It was observed that primary cultures derived froM

control rabbit vascular smooth muscle synthesized approxi­

mately 50 % type I collagen and 50% type III collagen (Table

3) . Analogous tissue in the arteriosclerotic group synthe­

sized 63% type I collagen and only 37 % type III. Gay et al.

(1975) by immunofluorescent techniques have shown that

type III occurs in human aorta, to the exclusion of t ype I

collagen, in the area immediately underly ing the internal

elastic lamella. Type III concentration in the media, rel­

ative to type I, diminishes toward the adventitia. McC ullagh

and Belian (1975) reported 70% t ype I and 30% type III in a

nondiseased normal human aorta and a shift in the extracted

collagen ratio favoring type I in arteriosclerosis. The

data reported here confirms that observation. There was

59

considerable sample variation in both expe r imental groups

with regard to the absolute amount collagen s yn thesis; how­

ever, this variation was reduced when the values were ex­

pressed per ug. of DNA. Fritz et al. (1975) reported that

in a similar s y stem there was a high degree of correlation

between protein and DNA synthesis. The fact that both types

of collagen increased in the arteriosclerotic groups, com­

?ared to controls, suggests a general activation of the

pathway for collagen synthesis specific for type I synthe­

sis in agreement with previous findings (Fuller et al.,

1976) .

The daughter cells growing in the tissue culture sys­

tem proved to be more efficient in producing labelled

collagen. In these cultures as much as 20% of the radio-

active protein released into the medium was collagenase di­

gestible; however, an average of about 10% was collagenase

digestible. It was still necessary to add carrier collagen

to the extracted 3H-proline labelled collagen in order to

visualize the characteristic collagen bands after separat ion

on po l yacrylamide gel s stained with Coomassie blue. The

total disappearance of radioactivity from t~e collagen pro­

tein bands by prior treatment of the sample with purified

clostridial collagenase (protease-free) consistentl y con­

firmed the identity of these characteristic bands.

Daughter cells derived from control aorta were s ynthe ­

sizing 60% type I and 40% t ype III collagen at the end of

their 4th passage (Table 4). Cells derived from arterio­

sclerotic aorta at the same passage and density (5 X 10 6/

75 cm 2 ) synthesized 80% type I and 20% type III collagen

The synthesis of collagen t ype I / ug. DNA was the same in

each g roup. The synthesis of type III was significantly

(p <0.05) decreased in the arteriosclerotic groupl however,

this decrease was not enough to produce a significant

(p <0.05) difference in total collagen s ynthesized b y the

experimental group of smooth muscle cells. The signifi­

cant decrease in type III s ynthesis was confirmed by CMC­

chromatography (Figure 5). As mentioned in the Results

section there was variation in the ratio of collagen types

determined by the different analytical methods for the cell

culture system. However, there was no observed disagree­

went in the ratio of these types s ynthesized by the organ

culture system. This may be due to the differential influ­

ences in each method by small amounts of aberrant or un­

natural forms of collagen (i.e. [ o<l (I)] trimer) that could

be intrinsic to the proliferating cell culture system of

daughter cells. No radioactivity above background could

be detected in the gel regions corresponding to the dimer

forms of collagen (pl,l orft 1 , 2 ) at this level of BAPN

supplement. It wa s determined by preliminary eA'Periments

that twice the normal concentration of BAPN was required

60

to inhibit cross-linking of collagen o<.-chains. These cell

lines were reported to be exceptionally rich in lysyl oxi­

dase activity (Herbert Kagan, personal conununication).

Rhoads and Miller (1978) have recently isolated and

characterized two new types of collagen chains designated

A and B. Based on their composition both chains resemble

collagen chains isolated from basement membrane. McCullagh

and Sweet (1978) have indicated that smooth muscle cells in

culture synthesize collagen A and B chains which are minor

components of the cellular expression of collagen synthe­

sis ( < 2 %) (Brown et al., 1978). Thus, these chains are

not synthesized in large enough quantity to interfere in

the detection of a s ignificant shift in the ratio of type I

and type III collagen. However, they may account for some

of the variability between the electrophoretic and chroma­

tographic results, since the A chain migrates in the same

position as the ~1-chain on sodium dodecyl-sulfate poly­

acrylamide gels.

The calculation of the relative ratios of collagen

types as auantitated here could be carried out by several

means. In this investigation the ratio of the radioactivity

for o<l: 0(2 varied from 1. 8 to 2. 3 and was considered to be

within acceptable limits of sensitivity for the anal ytical

techniques. Type I collagen was quantitated by measuring

the total cpms in the ~l band plus and ~2 band in unreduced

samples. However, if one were to strictly adhere to the

61

62

theory that ~l: ~2 must be in a 2:1 ratio then it is possible

that in some samples as much as 10% type I trimer [~l(I) J 3

could be present. It has been shown that when chick chondro-

cytes are grown in the presence of BUdR for a few days,

collagen biosynthesis will switch from cartilage specific

type II collagen [~l(II) J3 to a mixture of type I collagen 2

[o<l (I)] 2.o< and type I trimer [o<l (I)] 3 (Mayne et al., 19 75) .

It has been suggested that the formation of type I trimer is

associated with the shift in gene expression responsible for

a change in the type of collagen produced (Mayne et al.,

1976). It is evident from this study that atherogenic fac-

tors that modify the growth characteristics of vascular

smooth muscle cells also modify t::.e gene expression for dif -

ferent collagen types.

By the tenth passage in culture SMC derived from both

control and arteriosclerotic groups were synthesizing ex-

elusively type I collagen. Considerable difficulty was ex-

perienced in carrying SMC in the control group to the 10th

passage, due to an apparent decrease in plating efficiency

and growth rate, suggestive of senescence. Coliagen hetero-

geneity was measured in only two flasks of control cells at

the 10th passage and found to be about 90% t ype I for each.

The effect of time in culture on the s y nthesis of type I

collagen can be explained b y what has been reported regard-

ing "collagen switching." Cellular senescence appears to

be delayed in the arteriosclerotic cell group, which is

consistent with the other indication of dedifferentiation

mentioned above . The SMC in the control group proliferate

at a slower rate which suggests that their growth req~ire­

rnents are more stringent than those for the cells in the

arteriosclerotic group . The SMC in the control group may

be more dependent than SMC in the arteriosclerotic group,

63

on species-specific growth factors not present in the heter­

ologous , ~eat inactivated, fetal calf serum . A decrease in

the requirement for multiplication stimulating factors in

serum has been observed in viral transformed chick embryo

fibroblasts (Smith et al., 1971) . These types of factors

could modulate the onset of senescence.

64

CONCLUSI0NS

1) . The migration of smooth muscle cells from aorta extracts

is more rapid in tissue derived from arteriosclerotic

lesions than from control aorta. This supports the hy­

pothesis that there is a greater population of these

cells with enhanced migratory potential in the arterio-

sclerotic lesions. It is suggested that these modified

smooth muscle cells would similarly be more attracted

to the injured intima in the in vivo situation.

2). Daughter cells from arteriosclerotic smooth muscle pro­

liferate in culture at a higher rate than their counter-

parts from normal tissue. These modified smooth

muscle cells resemble transformed cells which are

generally considered to have exaggerated or higher

growth kinetics than untransformed cells.

3) The population of smooth muscle cells derived from

arteriosclerotic aortas contain cells which are

phenotypically different fro!"\ smooth muscle cells

from control aorta with respect to collagen heterogen­

eity. This disease related change in collagen hetero­

geneity has been reported in vivo and now has been

shown to be characteristic in the genome of daughter

cells maintained in vitro.

4). Derepression of collagen type I synthesis has been

linked with cellular processes associated with aging,

scars and transformation as observed for chondrocy tes

in culture. It appears that arteriosclerosis en ­

hances this derepression process for vascular smooth

muscle cells permitting the accumulation of fibrous

collagen components resembling scar tissues which be­

come fibrous vascular plaques.

5). These data also support a clonal selection hypothesis.

65

If a cell only synthesizes one type of collagen at a

time, tissues synthesizing more than one type of colla­

gen have more than one, type-specific 909ulation, of

coll~gen producing cells. The numerous population

doublings and subsequent subculturing procedures may

favor for one subpopulation of SMC (e . g . type I colla­

gen producing) and not the other subpopulation (e . g .

type III collagen producing) .

66

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75

VITA

Douglas O. Fisher was born on May 15, 1948 in Pittsburgh

Pennsylvania. Dr. Fisher entered the University of Rhode

Island in 1966 and received his Bachelor of Science Degree

in Pharmacy in 1971. In 1973 he completed a residency pro­

gram in Hospital Pharmacy at Roger Williams General Hospital,

Providence, Rhode Island. Dr. Fisher then continued his

education at the University of Rhode Island in the Department

of Pharmacology and Toxicology, completing the requirements

for Master of Science degree in March 1976 and Doctor of

Philosophy in March 1979.

Throughout his graduate training Dr. Fisher worked as

a technician for the University of Rhode Island Crime Labor­

atory. He is a member of Sigma Xi research society, Ameri­

can Society of Hospital Pharmacists and Rhode Island Society

of Hospital Pharmacists.

Dr. Fisher has accepted a position as a toxicologist

in the Department of Drug Safety Evaluation at Pfizer Inc.,

Groton, Connecticut.

Dr. Fisher is married to the former Kathleen N. Osborne

of Warwick, Rhode Island.